1. Field of the Invention
The present invention relates to a novel seven-pass transmembrane receptor protein found in immature dendritic cells and a DNA encoding the same. More particularly, the present invention is concerned with a human seven-pass transmembrane receptor protein having the amino acid sequence of SEQ ID NO:2 and a DNA encoding the same. By the use of the seven-pass transmembrane receptor protein and the DNA encoding the same which are provided according to the present invention, it has become possible to screen a substance which can be used for treating or preventing diseases mediated by the functions of dendritic cells, and to provide a method and a reagent for the diagnosis of such diseases. Also, the present invention is concerned with a replicable recombinant DNA which comprises a replicable expression vector and, operably inserted therein, the above-mentioned DNA; a cell of a microorganism or cell culture (transformant), which is transformed with the above-mentioned replicable recombinant DNA; a seven-pass transmembrane receptor protein which is produced on the cell surface of the above-mentioned transformant; a method for screening a ligand which binds to the above-mentioned seven-pass transmembrane receptor protein, and a method for screening a substance which inhibits the ligand from binding to the seven-pass transmembrane receptor protein; and an antibody which binds to the above-mentioned seven-pass transmembrane receptor protein. The present invention is also concerned with a method for the diagnosis of an inflammatory disease, which comprises determining the amount of the seven-pass transmembrane receptor protein expressed in human leukocytes.
2. Prior Art
When the living body of an organism suffers a disorder, such as an infection or inflammation, the cells at the site of the disorder release various protein factors, thereby transmitting the information of the disorder to the cells participating in the biological defense mechanism. In the case of infections, leukocytes, especially granulocytes (for example, neutrophils) and macrophages, have the function to trigger the biological defense mechanism. Like the granulocytes, dendritic cells (i.e., star-shaped cells having outwardly extending dendrites wherein the star-shaped cells are found in lymph nodes and germinal centers) are derived from myeloid hematopoietic stem cells, and play an important role in immunity and inflammation, especially in antigen presentation.
A macrophage is one type of the antigen presenting cells and it presents an antigen to activated T and B cells (Sornasse et al., J. Exp. Med., 175, 15-21, 1992). However, the activation of helper T cells depends on the antigen presentation by the dendritic cells. It is considered that the dendritic cells capture antigens in peripheral blood, and move to lymph, and mature in lymph nodes. It has been reported that both macrophages and dendritic cells at any stage of maturation can present an antigen to the activated T cells; however, only matured dendritic cells can sensitize naive T cells (Mehta-Damani et al., J. Immunology, 153, 996-1003, 1994). Dendritic cells are derived from hematopoietic stem cells, but precursor dendritic cells and immature dendritic cells are found in blood and lymph, and fully matured dendritic cells are found in spleen and lymph nodes. In general, the immature dendritic cells have the high ability to take in an antigen, and the higher the degree of the maturation of the dendritic cells, the higher their ability to perform antigen presentation becomes. The dendritic cells presenting an antigen express large amounts of MHC (Major Histocompatibility Complex) class I proteins and class II proteins.
As described above, the dendritic cells participate in immunity and inflammation in the living body and serve to protect the living body by a beneficial immune response. However, in some cases, the dendritic cells also cause an undesirable immune response (such as an autoimmune response) and an undesirable inflammatory reaction. Therefore, it is considered that if a method for regulating the functions of the dendritic cells can be found out, it will become possible to treat infections and tumors by causing a beneficial immune response and to treat autoimmune diseases and the like by suppressing an adverse immune response.
The functions of the dendritic cells, such as proliferation, differentiation, activation and chemotaxis, are regulated by various receptor proteins expressed by the dendritic cells. A receptor is a protein which is present on the surface of a cell and which binds, with high affinity, to a specific substance (a signaling molecule) which is found on the surface of another cell or in body fluid. When the signaling molecule binds to the receptor, the binding (which is an extracellular event) is converted by the receptor into an intracellular signal to thereby cause a cellular response (Alberts, Bruce et al. eds., xe2x80x9cMolecular Biology of the Cellxe2x80x9d, 2nd ed., Garland Publishing, Inc., pp. 681-726, 1989). The substance which binds to a receptor is generally called a xe2x80x9cligandxe2x80x9d. It is conceived that a substance which affects the functions of a receptor expressed by the dendritic cells can be used for negative or positive regulation of the dendritic cell functions thereby treating diseases caused by excess functioning or insufficient functioning of the dendritic cells. Examples of substances which affect the functions of a receptor expressed by the dendritic cells include a substance which binds to the receptor to thereby stimulate the cells, a substance which binds to the receptor to thereby inhibit the receptor from being stimulated by other ligands, and a substance which binds to the receptor to thereby inhibit a stimulation by other ligands from being transduced into the cell.
Examples of various receptors known in the art include cytokine receptors, EGF (Epidermal Growth Factor) receptors and seven-pass transmembrane receptors (xe2x80x9cThe Leukocyte Antigen Facts Bookxe2x80x9d, Academic Press Inc., 38-49, 1993), and the functions of these receptors are diverse. A seven-pass transmembrane receptor, which is one of the above-mentioned receptors, is also called a xe2x80x9cG-protein coupled receptor (GPCR)xe2x80x9d or a xe2x80x9crhodopsin-type receptorxe2x80x9d. Studies on the seven-pass transmembrane receptors have begun only recently, and it is believed that a large number of still unknown seven-pass transmembrane receptors exist.
An explanation is made below taking leukocytes as an example. Examples of receptors which have been identified as the seven-pass transmembrane receptors present in leukocytes include receptors which bind to anaphylatoxins, receptors which bind to chemokines and receptors which bind to PAF (Platelet-Activating Factor). An anaphylatoxin receptor participates in the functions of neutrophils and macrophages, such as the production of active oxygen, chemotaxis and activation of cell adhesion (Bouley, F. et al., Biochemistry, 30, 2993-2999, 1991). Further, the below-described observation has been reported in connection with IL-8 (Interleukin 8) receptor, which is one of chemokine receptors. When an inflammation inducer is intra-abdominally administered to a mouse deficient in a homologue of IL-8 (Interleukin 8) receptor, various phenomena are observed, such as a decrease in neutrophil infiltration, an onset of neutrophilia (phenomenon wherein activation and proliferation of neutrophils occur, but infiltration by neutrophils does not take place), and an increase in granulocytes and plasma cells in bone marrow and lymph nodes (Hisashi IIZASA and Kouji MATSUSHIMA, xe2x80x9cRinsho Men-eki (Clinical Immunology)xe2x80x9d, 28, 731-737, 1996). Among the substances which act on the receptors, the substances which are considered to have the possibility of being useful as pharmaceuticals include substances (such as IL-8 and MCP-1 (Monocyte Chemotactic Protein 1)) which bind to the receptors to thereby stimulate cells, and the substances (such as IL-8 mutants) which bind to the receptors to thereby inhibit the receptors from being stimulated by other ligands (Howard, O. M. Z et al., TIBTECH, 14, 46-51, 1996). However, in many cases, a single receptor binds to any of a plurality of signaling molecules, and a single signaling molecule binds to any of a plurality of receptors. Therefore, for developing a treatment for a disease, knowledge of only signaling molecules participating in the disease is insufficient. For example, fourteen different receptors are known to bind to the same signaling molecule called serotonin. These fourteen different receptors for serotonin include not only seven-pass transmembrane receptors, but also an ion channel-type receptor having a signal transduction pathway which is completely different from those of the seven-pass transmembrane receptors. In addition, with respect to each of the fourteen receptors, a compound which specifically binds thereto is known (1996 Receptor and Ion Channel Nomenclature, Supplement 1-81 Trends Pharmacol. Sci., 1996), and, therefore, studies have been made for individually using these receptors for the treatment of different diseases. Further, in the case of chemokines, it is well known in the art that a single signaling molecule (one type of chemokine) reacts with any of a number of different receptors and, at the same time, a single receptor reacts with any of a number of different signaling molecules (different types of chemokines) (Power, C. A. et al., Trends Pharmacol. Sci., 17, 209-213, 1996).
As apparent from the above, even when a disease is caused by a single signaling molecule, the signaling molecule binds to any of a number of different receptors which are present on different types of cells. Therefore, for specifically regulating the functions of a specific type of cell which is causative of the disease, it is important to specify the receptor which is expressed on the cell rather than specifying the signaling molecule which acts on the cell. For example, in the case of a chemokine (which is one of signaling molecules), no single type of leukocyte which reacts with the signaling molecule RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) can be specified because there are different types of leukocytes which are reactive with RANTES. On the other hand, eosinophils (one type of leukocytes) specifically express the chemokine receptor CCR3 (Cxe2x80x94C Chemokine Receptor 3), and, hence, a method for specifically regulating eosinophils can be searched by using the receptor CCR3 (Howard, O. M. Z. et al., TIBTECH, 14, 46-51, 1996).
The human receptors and the receptors in other species are known to exhibit different reactions with m the same compound (see, for example, Marleau, S. et al., J. Immunol. 157, 4141-4146, 1996). For example, some substances which activate the human receptors are known to inhibit the activation of the receptors in other species. Further, some receptors are known to act as a receptor for viruses during viral infection (see, for example, Choe, H. et al., Cell 85, 1135-1148, 1996), and it is also known that a molecule which binds to such a receptor prevents the viral infection (see, for example, Bleul, C. C. et al., Nature, 382, 829-833, 1996). In this case, it is important to identify the receptor expressed by the cells wherein the receptor is used by the viruses for infection. It is also known that specific types of viruses can complete infection only when the virus binds to a receptor in a specific species.
There are signaling molecules (e.g., PF4 and HCC1 which are chemokines) which are presumed to bind to receptors which are seven-pass transmembrane receptors, wherein, however, the receptors have not yet been identified (Premack, B. A. et al., Nature Medicine, 2, 1174-1178, 1996; and Loetscher, M. et al., J. Exp. Med., 184, 963-969, 1996). Especially, with respect to the chemokines, a number of still unknown chemokines are presumed to exist (Howard, O. M. Z. et al., TIBTECH, 14, 46-51, 1996), and, therefore, a number of receptors which bind to the unknown chemokines are also expected to exist.
As in the case of the leukocytes described above, not all of the receptors for the molecules which act on the dendritic cells have been found out, and a number of seven-pass transmembrane receptors are expected to exist on the dendritic cells. As an example of seven-pass transmembrane receptors present on mature dendritic cells, ChemR23 has been reported (Samson, M. et al., Eur. J. Immunol., 28, 1689-1700, 1998). A method for regulating the functions of dendritic cells and ultimately for regulating diseases can be established when it becomes possible to obtain various receptors expressed in the dendritic cells at different stages of differentiation and to obtain substances which affect the functions of each of the obtained various receptors.
With respect to the endogenous substances which act on the seven-pass transmembrane receptors, various substances are known for various receptors. For example, glutamic acid and dopamine (which are both physiological amines) bind to the glutamic acid receptors and the dopamine receptors, respectively. Further, neuropeptide Y and endothelin (which are both peptides) bind to the neuropeptide Y receptors and the endothelin receptors, respectively (Watson, S. and Arkinstall, S., xe2x80x9cThe G-protein Linked Receptor Facts Bookxe2x80x9d, Academic Press Inc., 1994). Such endogenous substances include both substances (such as chemokines and PAF) which are known to act on the leukocytes, and substances which do not act on the leukocytes.
A substance which activates the seven-pass transmembrane receptor, irrespective of whether natural or artificial, causes various changes in the intracellular signals, and the changes caused in the intracellular signals depend on the states of the substance, the seven-pass transmembrane receptor itself and the cell expressing the receptor. Examples of such changes in the intracellular signals include an increase and decrease in the intracellular CAMP concentration, an increase in the inositol phosphate concentration, and an increase in the intracellular calcium concentration (Watson, S. and Arkinstall, S., xe2x80x9cThe G-protein Linked Receptor Facts Bookxe2x80x9d, Academic Press Inc., 1994). Methods for measuring each of these changes have been developed. Therefore, by measuring the changes exemplified above, it becomes possible to determine whether a particular substance activates a particular seven-pass transmembrane receptor or inhibits the activation of a particular seven-pass transmembrane receptor. Further, methods for observing physiological phenomena (such as cell proliferation, changes in gene expression, and chemotaxis) which are caused by the binding of the substance to the seven-pass transmembrane receptor are also known in the art, and these phenomena can be used as the indices for determining whether a particular substance activates the seven-pass transmembrane receptor or inhibits the activation of the receptor. As apparent from the above, there are a wide variety of methods for identifying a substance which acts on the seven-pass transmembrane receptor. However, it should be noted that for using such methods for obtaining substances useful as pharmaceuticals for humans, it is first required to obtain a human seven-pass transmembrane receptor protein.
Likewise, if there can be obtained substances which individually act specifically on different types of seven-pass transmembrane receptors (for example, the chemokine receptors), such substances may possibly lead to development of novel pharmaceuticals which each selectively suppress a specific inflammatory reaction and the like.
An explanation is made below taking chemokines as an example. The chemokines and the chemokine receptors regulate the chemotaxis of each of various different leukocytes. Therefore, it is considered that a particular leukocyte expresses a particular chemokine receptor. Actually, there are reports that CCR5 is expressed by Th1 cells and CCR4 is expressed by Th2 cells (Loetscher, P. et al., NATURE, 391, 344-345, 1998; and Bonecchi, R. et al., J. Exp. Med., 187, 129-134, 1998), and the chemokine receptors are considered to participate in the selection of specific cellular and humoral immune responses following an antigen non-specific inflammation. In addition, chemokines, such as CXC and CC chemokines, which act mainly on neutrophils and monocytes are called xe2x80x9cInflammatory chemokinesxe2x80x9d because these chemokines play an important role in acute or chronic inflammatory reactions. The detection of inflammatory diseases, the diagnosis of the severity of the diseases and the evaluation of the progress of treatment become possible by conducting studies on the receptors for the inflammatory chemokines, for example, by conducting the analysis of the expression of the chemokine receptors in the peripheral blood.
It is considered that the analysis of the receptors will possibly lead to the development of novel methods for the diagnosis and treatment of inflammatory diseases and the like of which the pathogenesis is not known. For example, rheumatism is a cryptogenic, systemic inflammatory disease exhibiting recurrent erosive arthritis as a major symptom thereof while giving impairment to multiple organs. Rheumatism progresses chronically while repeating remission and exacerbation m and leads to the destruction and deformation of joints, and finally causes the functional disorder of motorium. At present, the important factor of the treatment of rheumatism is to diagnose rheumatism at a stage as early as possible and suppress the rheumatic inflammations as soon as possible and as much as possible so as to prevent the occurrence of symptoms, such as joint damages, which are impossible to cure.
As apparent form the above, it is very important to start the treatment of rheumatism at an early stage. During the treatment of rheumatoid arthritis (RA) by the conventional pyramid method (Smyth, C. J., Postgrad. Med., 51, No. 6, 31-39, 1972), the treatment using only a nonsteroidal anti-inflammatory drug (NSAID) is conducted for 3 to 6 months and, then, only after determining that the patient is actually suffering from rheumatoid arthritis (RA), a disease-modifying anti-rheumatic drug (DMARD) is administered to the patient. When RA progresses during the treatment with NSAID, resulting in an increase in the number of arthritic joints, the effect of the DMARD decreases markedly and the prevention of bone damages becomes difficult. Therefore, early diagnosis of RA is necessary for the administration of DMARD to be started at an early stage before the occurrence of bone damages. According to the current criteria for the diagnosis of RA (Arnett, F. C. et al., The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis, Arthritis Rheum. 31, 315-324, 1988), it is difficult to diagnose RA at the early stage, and at least 6 weeks are necessary for the diagnosis. In the clinical observations, the early RA patients exhibit significantly high values with respect to the sedimentation rate of erythrocytes (wherein erythrocyte sedimentation is a non-specific reaction which is promoted by the destruction and inflammation of tissues) and the amount of CRP (C-reactive protein) which is an acute reaction product which rapidly increases upon the occurrence of the destruction and inflammation of tissues. However, none of these reactions are RA specific, so that they cannot be used for distinguishing RA from other diseases, such as collagenosis. In addition, there are cases of early RA wherein the erythrocyte sedimentation rate is normal, but bone damages are observed by X-ray examination. Further, 71% of the early RA patients who are within 1 year from the onset of RA are positive with respect to the rheumatoid factor, but the rheumatoid factor positive ratio among the RA patients who are within 6 weeks from the onset of RA is only 59%. No significant differences in the numbers of leukocytes and erythrocytes and hemoglobin level are observed between early RA patients (within 1 year from the onset) and early non-RA arthritis patients (Kuniomi YAMAMAE, xe2x80x9cIgaku no ayumi (Progress in Medicine)xe2x80x9d, 182, No. 9, 605-610, 1997).
Thus, although early diagnosis is necessary for the treatment of rheumatism, no diagnostic marker which is useful for the diagnosis of rheumatism has yet been established in the art. Therefore, the early diagnosis of rheumatism is difficult.
The present inventors have made extensive and intensive studies with a view toward solving the above-mentioned problems. In the course of the studies, the present inventors conceived that immature dendritic cells have a novel seven-pass transmembrane receptor and such a novel receptor is useful for searching pharmaceuticals which can regulate the functions of dendritic cells. First, by employing various methods, such as differential display method (for example, see Liang, P. et al., Curr. Biol., 7, 274-280, 1995), RDA method (Lisitsyn, N. et al., Science, 259, 946-951, 1993) and degenerative PCR method (Innis, M. A. et al., PCR Protocols, 39-53, 1990), the present inventors worked for obtaining a cDNA for a novel seven-pass transmembrane receptor protein expressed in the immature dendritic cells. In particular, with respect to the degenerative PCR method, more than 20 types of primers including those primers used in Example 1 described below were tested. As a result of such extensive and intensive studies, the present inventors successfully obtained a cDNA fragment of a novel seven-pass transmembrane receptor from dendritic cells, and subsequently succeeded in cloning the entire coding region of the obtained cDNA fragment. The present inventors designated the novel seven-pass transmembrane receptor as xe2x80x9cC5L2xe2x80x9d. Further, the present inventors prepared an expression system for the receptor protein encoded by the novel DNA. In addition, the expression of C5L2 in human tissues, leukocytes, leukemia cell lines and the like was examined by northern blotting and it was found that leukocytes, especially granulocytes, exhibit strong expression of C5L2 gene.
Since the structure of the novel seven-pass transmembrane receptor C5L2 protein is similar to those of receptors for chemotactic factors, such as chemokines, FMLP and C5a, studies were made on the possible use of the novel receptor in various fields, such as pharmaceuticals for various inflammatory diseases, and the diagnosis and treatment of various inflammatory diseases. Further, the present inventors considered that the receptor of the present invention should be useful for the diagnosis of inflammatory diseases, and hence made research into the relationship between rheumatism and the receptor C5L2. As a result, it was found that the seven-pass transmembrane receptor C5L2 is useful for the diagnosis of inflammatory diseases. The present invention has been completed, based on these novel findings.
Therefore, it is a primary object of the present invention to provide a human seven-pass transmembrane receptor protein which is advantageous for searching pharmaceuticals which can regulate the functions of dendritic cells.
It is another object of the present invention to provide a DNA encoding the above-mentioned seven-pass transmembrane receptor protein, a recombinant DNA obtained by operably inserting the DNA into an expression vector, and a cell of a microorganism or cell culture, transformed with this recombinant DNA.
Still another object of the present invention is to provide a method for screening a ligand which binds to the seven-pass transmembrane receptor protein, and a method for screening a substance which inhibits the ligand from binding to the seven-pass transmembrane receptor protein.
A further object of the present invention is to provide an antibody which binds to the seven-pass transmembrane receptor protein.
Still a further object of the present invention is to provide a method for the diagnosis of an inflammatory disease, which comprises determining the amount of the seven-pass transmembrane receptor protein expressed in human leukocytes.
The foregoing and other objects, features and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and the appended claims taken in connection with the accompanying drawings and sequence listing.
In SEQ ID NO:5, the 18th, 22nd and 24th nucleotides xe2x80x9cnxe2x80x9d represent inosine (i). This sequence is of a degenerative PCR primer designed based on the nucleotide sequences of conventional seven-pass transmembrane receptor proteins which are considered to participate in the proliferation of melanoma.
In SEQ ID NO:6, the 22nd and 28th nucleotides xe2x80x9cnxe2x80x9d represent inosine (i), and the 21st nucleotide xe2x80x9cnxe2x80x9d represents a, g, c or t. This sequence is of a degenerative PCR primer designed based on the nucleotide sequences of conventional seven-pass transmembrane receptor proteins which are considered to participate in the proliferation of melanoma.
SEQ ID NO:7 is of a synthetic primer used for constructing the-recombinant DNA containing C5L2 gene, wherein the primer has a sequence obtained by adding the spacer sequence xe2x80x9cggggxe2x80x9d and restriction enzyme HindIII recognition site xe2x80x9caagcttxe2x80x9d to the 5xe2x80x2-end of a 22-nucleotide sequence corresponding to the 1st (a) to 22nd (t) nucleotides of SEQ ID NO:1.
SEQ ID NO:8 is of a synthetic primer used for constructing the recombinant DNA containing C5L2 gene, wherein the primer has a sequence obtained by adding the spacer sequence xe2x80x9cgggaxe2x80x9d and restriction enzyme SacII recognition site xe2x80x9cccgcggxe2x80x9d to the 5xe2x80x2-end of a 20-nucleotide sequence corresponding to the 206th (c) to 225th (a) nucleotides of SEQ ID NO:4.
SEQ ID NO:9 is of a synthetic primer used in RT-PCR performed for amplifying C5L2 gene.
SEQ ID NO:10 is of a synthetic primer used in RT-PCR performed for amplifying C5L2 gene.
SEQ ID NO:11 is of a synthetic primer used in RT-PCR performed for amplifying G3PDH gene (glyceraldehyde 3-phosphate dehydrogenase).
SEQ ID NO:12 is of a synthetic primer used in RT-PCR performed for amplifying G3PDH gene (glyceraldehyde 3-phosphate dehydrogenase).